Prostaglandins (PGs) are potent biologically active compounds, known to act as local tissue hormones capable of triggering a multiplicity of physiological responses. In salivary submandibular and sublingual glands, PGs, especially those of the E series, have been demonstrated to regulate salivary flow and mucin secretion, and may play a role in the control of electrolyte concentrations in saliva. However, the cellular mechanism of PGEs action in salivary glands is poorly understood. The PI has recently characterized a specific PGE2 receptor in crude membranes of salivary submandibular glands. The binding of PGE2 to a specific receptor on cell membrane may be the initial step leading to its biological actions within the cells. To better elucidate the role of PGE2 receptor in salivary glands at the cellular level, it is important to purify, identify and characterize the receptor protein, as well as to investigate the intracellular signal transduction pathway mediated by PGE2 and its receptor. The specific aims of this proposed project are: Aim #1. Purification, identification and characterization of PGE2 receptor in acinar cells of salivary glands. Acinar cells from submandibular and sublingual glands will be isolated. Various subcellular membrane fractions will be prepared and analyzed or PGE2 receptor binding. The receptor-enriched subcellular fraction(s) will be used for receptor purification and identification. Aim #2. Study of PGE2 receptor-mediated signal transduction in acinar cells of salivary glands. Semipurified acinar plasma membrane will be prepared and used to study the receptor-coupled intracellular second messenger system, which involves the functional associations among PGE2, receptor and N protein in the adenylate cyclase system. In situ protein phosphorylation after receptor binding will also be examined. A temporal correlation between the in situ protein phosphorylation and the intracellular cAMP production under the influence of PGE2 in a dose- dependent manner will be investigated. Purification and identification of receptor protein in salivary gland acini will not only support the data of biochemical features of the receptor, but also be a necessary prerequisite for future receptor studies at both the cellular and molecular levels. The proposed study will also reveal important information regarding the intracellular processing of signal after the receptor binding. The Principal Investigator (PI) anticipates that this proposed research will pave the avenues towards a better understanding of the regulatory role played by PGE2 and its receptor in salivary glands, and expects to elucidate the mechanism underlie the links between the intracellular signals and the biological functions triggered by PGE2 in salivary glands.